In this subject the conceptual bases of the experimental methodologies that allow the extraction,

analysis, cloning and expression of nucleic acids are worked on. These methodologies are used with the aim of taking steps in the knowledge of the different cellular functions and to be able to apply this knowledge in the fields of Biology, Biomedicine and Biotechnology.

This optional subject is offered within the Specialty of Cellular, Molecular and Genetic Biology and is based on the knowledge acquired by students in basic subjects of Cellular Biology, Biochemistry, Genetics, Microbiology and Molecular Genetics. The contents that are worked on are integrated and related to various subjects of the area of ​​Genetics and other areas such as Cellular Biology and Microbiology. The material is basic for the professional performance of any Molecular Biologist.

SPECIFIC COMPETENCES



1.Acquire a current perspective of the methodological and technological strategies used in Molecular Genetics and in the molecular analysis of genomes.

2.Understand and recognize the applications of molecular techniques and the manipulation of genomes, in the field of research in Biology, Biomedicine and Biotechnology and in the Agricultural industry

3. Know and practice basic technical procedures that allow the student to become familiar with molecular analysis.



TRANSVERSAL COMPETENCES



1.Develop the capacity for analysis and synthesis and progress in critical reasoning and ethical commitment

2.Develop the capacity for organization and planning

3. Delve into teamwork

THEORY PROGRAM

INTRODUCTION

1.- Recombinant DNA: Definition and objectives. General system for gene analysis and manipulation. The historical development of recombinant DNA technology.



FUNDAMENTALS OF DNA, RNA AND PROTEIN ANALYSIS AND MANIPULATION

2.- Basic techniques for the analysis and manipulation of nucleic acids and proteins: DNA, RNA and protein extraction. Purification, quantification and electrophoresis. Probes. Hybridization of nucleic acids. Southern, Northern and Microarrays. Protein detection: Western, Immunohistochemistry and Proteomics. Gene editing using CRISPR/Cas9.

3.- Techniques for the in vitro amplification of nucleic acids: Description of the PCR technique. Components and conditions in the reaction. Primer design. Some applications. qPCR and RT-qPCR. Basic techniques for sequencing: Sanger and Pyrosequencing.



GENE MODIFICATION IN BACTERIA

4.- Cloning of DNA in bacteria: recombinant DNA in bacteria. Characteristics of the bacterial host. Types and characteristics of the cloning vectors. Systems of transformation in bacteria. Selection of transformants. Extraction and purification of plasmid DNA.

5.- Heterologous gene expression in bacteria. Expression vectors. Elements of the expression systems. Fusion genes. Purification and detection of proteins. Marker genes. Applications of the bacterial transformation.

6.- Genomic libraries massive sequencing technics. DNAseq and RNAseq.



GENE MODIFICATION IN BACTERIA EUKARYOTES

7.- General characteristics of DNA cloning in eukaryotes. Transient and stable transfection.

8.- Genetically modified plants: Gene transfer and gene editing in plants. Gene transfer systems. Types and characteristics of cloning vectors. Heterologous gene expression control systems. Applications.

9.- Genetic modification of mammalian cells: Characteristics of the host cells. System of gene transfer. Types and characteristics of cloning vectors in mammals. control systems expression of heterologous genes. Applications.

10.- Inactivation, silencing and editing of genes: Gene inactivation by homologous recombination. Site specific recombination and conditional gene knockout. Gene silencing by RNA interference (RNAi): antisense oligonucleotides, siRNAs and miRNAs. Gene editing using CRISPR/Cas9.

11.- Genetically modified animals: Generation of transgenic mice: knock-out and knock-in. System for expression control. Generation of other transgenic animals: nuclear transfer. Applications.

12.- Gene therapy: Ex vivo and in vivo and somatic vs germinal gene therapy. Human cell transfection systems. Use of gene therapy in genetic diseases and acquired diseases.





PROGRAM FOR LABORATORY PRACTICE



Cloning of the lambda phage genome in the pUC18 plasmid:



a) Digestion of the phage lambda genome and the pUC18 vector. Ligation



b) Transformation of competent bacteria with the ligation product and seeding in selective medium



c) Extraction and purification of recombinant plasmids



d) Identification of the cloned fragments by analysis of the size of the cloned fragment after digestion and PCR

The subject includes different teaching modalities. Theoretical concepts are worked on in the lectures. The classroom practice sessions are related to the application of theoretical content to the resolution of situations, with the realization of quantitative estimations for their later experimental application, with the interpretation of experimental results, etc. In seminar sessions, students work critically on scientific texts related to the applicability of the learned methodologies and their safety and perception social.

• Continuous Assessment System
• Final Assessment System
• Tools and qualification percentages:
  • Written test to be taken (%): 35
  • Multiple-Choice Test (%): 15
  • Realization of Practical Work (exercises, cases or problems) (%): 30
  • Team projects (problem solving, project design)) (%): 10
  • Exhibition of works, readings ... (%): 10

The evaluation system includes a final exam and other tests that are part of the continuous evaluation:



1) The final written test (50% of the mark) consists of test questions (15%) and questions to develop (35%). So that

the course can be approved, a minimum of 4.0 points (out of 10) will be required in each of the sections.



2) The written tests carried out in groups and that are part of the continuous evaluation include the document related to the experimental work carried out in the laboratory sessions (30%), problem solving theoretical and practical (10%) and the delivery of the report related to the work carried out in the seminar sessions (10%). The evaluation of group activities will be individualized depending on the level of commitment and involvement with the group work carried out. For the subject to be approved, a minimum of 4.0 is required points (out of 10) in each of the sections.



Waiving continuous evaluation requires an explanatory letter addressed to teachers during the first 9 weeks of the course.



During the development of the evaluation tests, the use of books will be prohibited, as well as devices or

telephone, electronic, computer, or other devices, by students. You are only allowed to bring your class notes

and calculator.



In any case of dishonest or fraudulent practice, the provisions of the ethics protocol from the university will be applied (Prevention of dishonest or fraudulent practices in assessment tests and assignments academics at the UPV/EHU).



The non-presentation to the final test will mean the resignation of the evaluation call and will be recorded as a No submitted.

The positive results of the continuous assessment obtained by the students during the course are kept. In case of

Negative results in the continuous evaluation, the final evaluation test will contribute 100% of the qualification of the

subject.



During the development of the evaluation tests, the use of books will be prohibited, as well as devices or

telephone, electronic, computer, or other devices, by students. You are only allowed to take notes and

calculator.



In any case of dishonest or fraudulent practice, the provisions of the ethics protocol from the university will be applied (Prevention of dishonest or fraudulent practices in assessment tests and assignments academics at the UPV/EHU).



Failure to submit to said test will mean the resignation of the evaluation call and will be recorded as a No

submitted.

THE TEACHING STAFF WILL PROVIDE THE STUDENTS WITH THE FOLLOWING MATERIAL:
A collection of problems will be used as basic material that will be delivered to the students with sufficient advance. The collection includes problems that will not be solved in the classroom and that the student must use as material for personal work.
The protocol of the practices and the necessary documentation for the realization of the seminars will be given with sufficient advance.
In the case of the practices protocol, the objectives of each activity are included. Also its rationale theory, their technical development and some questions that each student must answer during or after completion of the corresponding practice. It is mandatory to read the protocol before carrying out the corresponding practice since in the laboratory no question will be answered regarding the protocol or related to previous theoretical knowledge that should have been reviewed previously. As for the seminars, the supporting documentation corresponding to each session will be given.
All the necessary documentation will be available in the virtual classroom to support this subject.

Basic bibliography

- Wink M. (redactor)(2021) An introduction to Molecular Biotechnology: Fundamentals, Methods and Applications. 3rd.

edition. Ed. Wiley ISBN: 978-3527344147.



- Real MD, Rausell C, Latorre A(2017)Técnicas de ingeniería genética. Editorial Síntesis. ISBN: 978-84-9171-071-4.



- Klug WS, Cummings MR, Spencer CA, Palladino MA. Killian D (2019) Concepts of Genetics. 12th edition (978-

1292265322).



- Brooker RJ (2021) Genetics. Analysis & Principles. 7/e. McGraw Hill (978-1260240856)



- Goldberg M, Fisher JA,Hood L, Hartwell L (2021) Genetics. From Genes to Genomes. 7th edition. McGraw-Hill (978-

1260240870).



- Nicholl D.S.T. (2008) An introduction to Genetic Engineering. Cambridge University Press (3ª edición) ISBN-10:

0521615216.



-Primose SB, Twyman RM (2006) Principles of Gene Manipulation and Genomics. Wiley-Blackwell (an imprint of John

Wiley & Sons Ltd); 7th Edition . ISBN: 978-1405135443.



- Stephenson F (2012) Cálculo en Biología Molecular y Biotecnología. Guía de matemáticas para el laboratorio. 2ª ed.

Elsevier. ISBN 8490220913.

In-depth bibliography

- Krebs J, Goldstein E, Kilpatrick (2018) Lewin´s Genes XII; Jones and Bartlett Publishers, Massachussets. ISBN:
978-1284104493
- Geoffrey M. Cooper (2018) The Cell: A Molecular Approach. 8ª Ed. Sinauer associates. ISBN: 1605357073
- Pierce, B.A (2017) Genetics Essentials: Concepts and Connections.(4rd Ed.).W. H. Freeman and Co. ISBN:
1319107222

Journals

Nature
Science
Nature Review Genetics

Web addresses

https://ocw.ehu.eus/course/view.php?id=397